Remedying a contaminated environment using Pseudomonas cepacia or Corynebacterium species and Renobacter species FERM BP-5353 having dehalogenase activity

ABSTRACT

A process for remedying an environment contaminated with an aliphatic organochlorine compound which includes the use of Pseudomonas cepacia strain KK01 (FERM BP-4235) or Corynebacterium species (FERM BP 5102) and Renobacter species (FERM BP-5353). The first two microorganisms are capable of introducing an oxygen atom into the aliphatic organochlorine compound in order to convert the aliphatic compound to an epoxide. During protonization the epoxide is converted into a chlorinated organic acid. Renobacter species strain FERM BP-5353 decomposes chlorinated organic acids to substances naturally existing in nature. The chlorinated and/or halogenated acids include chloroacetic acid, dichloroacetic acid, trichloroacetic acid and dichloropropionic acid, etc. The polluted environments in which the processes may be carried out include the soil, ground water and waste water.

This application is a division of application Ser. No. 08/561,237 nowissued as U.S. Pat. No. 5,679,568 filed Nov. 21, 1995, now U.S. Pat. No.5,679,568.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a process for decomposing a pollutant using amicroorganism, particularly a process for decomposing a halogenatedorganic compound using a microorganism which has an activity todecompose the halogenated organic compound, to a process for environmentremediation, which can decompose a pollutant in the environment, such asground water, soil and so on, and to a microorganism capable ofdecomposing a pollutant and being used in the decomposition andremediation processes.

2. Related Background Art

The by-products resulting from the disinfection process of city waterhave been a serious concern since the U.S. Environmental ProtectionAgency reported on them in the 1970s. In Japan also, where chlorinationof city water is compulsory, the by-products such as trihalomethanes,halogenated acetic acids, halogenated acetonitriles and halogenatedketones have been detected in city water, and has become a great problembecause of their liver toxicity and mutagenicity. Particularlyhalogenated organic acids, for example halogenated acetic acids such aschloroacetic acid, dichloroacetic acid, trichloroacetic acid andbromoacetic acid have been designated as environment surveillance itemsin Japan since 1993, attracting a great deal of attention as a newproblem. Details are reported in "Analytical Methods for RevisedStandards of Water Quality and Environment" in the Proceedings of the23rd Seminar of Water Environment Society of Japan (IncorporatedAssociation of Water Environment Society of Japan), November 1993,pp.55-64.

These halogenated organic acids cannot be degraded by aeration. As oneof the countermeasures, for example, biodegradation treatment such as abioreactor is very useful because treatment can be conducted under mildconditions and is relatively low in cost.

Microorganisms having a decomposing activity of halogenated organicacids have been studied, for example, molds such as Trichoderma,Acrostalagmus, Penicillium and Clonostachys, and bacteria such asPseudomonas, Arthrobacter, Rhizobium, Agrobacterium, Bacillus,Alcaligenes, Nocardia, Micrococcus, Achromobacter and Moraxella(Protein, Nucleic Acid and Enzyme (1984), vol.29, p.101-110). Adachireported that an unidentified strain OS-2 has an enzyme which candecompose chloroacetic acid, bromoacetic acid and iodoacetic acid toabout the same extent, and OS-2 can also decompose dichloroacetic acidthough to half the extent of the above compounds (Proceedings of OsakaPrefectural Institute of Public Health, "Public Health" No. 30, p.89(1992)). Furthermore, decomposition of halogenated organic acids havingtwo to six carbons was studied using dehalogenase extracted fromPseudomonas putida NCIMB 12018 and immobilized on carboxymethylcellulose or thioglycolic acid. (European Patent No. 179603).

The relation between dehalogenating enzymes for decomposing halogenatedorganic acids and their genes has been studied using Pseudomonas putidaAJ1 strain (J.Gen. Microbiol., 138, p.675 (1992)), Pseudomonas cepaciaMBA4 strain (J.Biochem., 284, p.87 (1992)) and Pseudomonas sp. CBS3strain (Biol. Chem. Hoppe-Seyler., 374, p.489 (1993)).

All these studies are done on the enzyme level, and not on the actualbehavior of the microorganisms in the polluted waste water. Concerningthe microbial decomposition of halogenated organic acids, onlyXanthobacter autotrophicus GJ10 strain (Appl. Biochem. Biotechnol., 40,158 and 40, 165 (1993)) has been studied.

Japanese Patent Laid-Open Application No. 4-64544, disclosesdehalogenase extracted from Pseudomonas which decomposes D- andL-chloropropionic acid into lactic acid. This is, however, also a studyat the enzyme level.

SUMMARY OF THE INVENTION

The present invention has been made to solve the above mentionedproblems and provides a biodegradation process of halogenated organicacids and a process for remedying environment using the process.

Another object of the present invention is to provide a biodegradationprocess giving substantially complete decomposition of aliphaticorganochlorine compounds and a process for remedying environment usingthe process.

Another object of the present invention is to provide a new microbialstrain which is suitably used in the biodegradation process ofhalogenated organic acids and aliphatic organochlorine compounds and inthe environment remediation using the process.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the decomposition of dichloroacetic acid bystrain AC.

FIG. 2 is a graph showing the decomposition of chloroacetic acid bystrain AC.

FIG. 3 is a graph showing the decomposition of trichloroacetic acid bystrain AC.

FIG. 4 is a graph showing the decomposition of bromoacetic acid bystrain AC.

FIG. 5 is a graph showing the decomposition of chloropropionic acid bystrain AC.

FIG. 6 is a graph showing the decomposition of 2,2-dichloropropionicacid by strain AC.

FIG. 7 is a graph showing the decomposition of dichloroacetic acid insoil by strain AC.

FIG. 8 is a graph showing the decomposition of halogenated acetic acidsin soil by strain AC.

FIG. 9 is a graph showing the decomposition of chloropropionic acids insoil by strain AC.

FIG. 10 is a graph showing the decomposition of 2,2-dichloropropionicacid in soil by strain AC.

FIG. 11 is a graph showing the results of Example 10.

FIG. 12 is a graph showing the results of Comparative Example 1.

FIG. 13 is a graph showing the results of Example 11.

FIG. 14 is a graph showing the results of Comparative Example 2.

FIG. 15 is a graph showing the results of Example 12.

FIG. 16 is a graph showing the results of Comparative Example 3.

DETAILED DESCRIPTION OF THE INVENTION

The above-mentioned objects will be accomplished by the presentinvention described below.

Searching for microbial strains having halogenated organicacid-decomposing activity, the present inventors obtained a newmicrobial strain which can decompose a high concentration of halogenatedorganic acids, from the soil of Kanto loam layer in Japan. The presentinventors have found a process for decomposing halogenated organic acidsin aqueous medium by exposing it to the above strain.

The complete taxonomic description of FERM P-14641, now identified asFERM BP-5353, is as follows:

    ______________________________________                                        A.       Morphology                                                                  Gram stain:  negative                                                               cell size and shape:                                                                       C and/or S shape rod                                           1.0-2.0 μm in                                                             length                                                                       0.2-0.5 μm in width                                                        motility: none                                                             color of colony:  white to cream                                          B. Growth in culture media                                                    BHIA: good                                                                     MacConkey:  poor                                                             C. Optimum growth temperature: 25-35° C.                               D. Physiological characteristics                                                  Aerobic or anaerobic:  aerobic                                               TSI (slant/butt): alkali/alkali, H.sub.2 S (-)                               Oxidase:  positive                                                            Catalase: positive                                                        ______________________________________                                    

In view of the characteristics mentioned above, the present strain issuitably classified into Renobacter sp.

As is evident from the following Examples, the present strain has anexcellent activity to decompose halogenated organic acids. Since strainscapable of decomposing halogenated organic acids had not been known inRenobacter species, the present strain was acknowledged as a new strain,and named Renobacter sp. AC. It was deposited in National Institute ofBioscience and Human-Technology, Agency of Industrial Science andTechnology (Deposition No.: FERM BP-5353).

Strain AC is very unique in its growth style in addition to the cellshape (S-shaped). It grows forming cell aggregates secreting a certainkind of polymeric material(s) not identified yet. From the microscopicviewpoint, this characteristic allows strain AC to rapidly form its ownhabitat and to become a prior species in drainage, liquid wastes, riversand lakes where various kinds of microorganisms exist and beadvantageously used for decomposing halogenated organic acids in suchplaces.

Although the present strain can be cultured in a natural complete mediumsuch as 2YT and LB, it can also be cultured in an inorganic salt medium,for example M9, supplemented with a small amount of yeast extract as anutrient.

The composition of M9 is as follows:

Na₂ HPO₄ : 6.2 g

KH₂ PO₄ : 3.0 g

NaCl: 0.5 g

NH₄ Cl: 1.0 g

in 1 litter of medium (pH 7.0).

Culture can be carried out under aerobic conditions and in either liquidor solid medium. The temperature for culture is preferably about 30° C.

It is evident that any mutant spontaneously or artificially derived fromthe present strain is included in the scope of the present invention solong as it has a good decomposing activity for halogenated organicacids. Accordingly, examples using such a mutant are also included inthe scope of the present invention.

In one embodiment of the present invention, decomposition of halogenatedorganic acids can be carried out by bringing in contact the aboveRenobacter sp. AC with halogenated organic acids in an aqueous mediumsuch as liquid wastes and the like. It can be carried out by culturingthe microorganism in the aqueous medium containing halogenated organicacids, or by adding the aqueous medium to the culture of themicroorganism, using various methods such as a batch method, asemi-continuous method and a continuous method. The microorganism may bein a state semi-immobilized or full-immobilized to a suitable carrier.As described above, the present strain can be easily and advantageouslyimmobilized because it grows into a mass secreting a polymericmaterial(s) by itself.

In another embodiment of the present invention, decomposition ofhalogenated organic acids can be carried out by bringing in contactRenobacter sp. AC with halogenated organic acids in soil. It can beconducted by culturing the strain in the soil containing halogenatedorganic acids or by mixing the polluted soil into the culture of thestrain.

This process can be used for remedying soil both in the closed systemand the open system, and the process is carried out by various methodssuch as batch method, semi-continuous method and continuous method. Themicroorganism may be in a state semi-immobilized or full-immobilized toa suitable carrier. As described above, the present strain can be easilyand advantageously immobilized because it grows into a mass secreting apolymeric material(s) by itself.

The present inventors also found a process for decomposing aliphaticorganochlorine compounds, for example, trichloroethylene (hereafterreferred to as TCE) into substances naturally existing in nature and aprocess for remedying environment using the above process, applying theabove-mentioned process for decomposing halogenated organic acids.

Recently, environmental pollution with aliphatic organochlorinecompounds which are harmful to living bodies and hardly decomposable hasbecome a serious problem. Particularly, it is considered that the soilin a manufacturing area of high technology industry is polluted withorganochlorine compounds such as tetrachloroethylene (PCE),trichloroethylene (TCE) and dichloroethylene (DCE), and the pollution isexpanding in a wide range. Actually, such organochlorine compounds weredetected by environmental research and reported frequently. It is saidthat these aliphatic organochlorine compounds in soil are dissolved inthe rain water to flow into the groundwater, thus the pollution expandsto the surrounding area. These compounds are suspected ofcarcinogenicity, and very stable in environment. Accordingly it has beena serious social problem that ground water, which is utilized as asource of drinking water, is polluted with such compounds.

In regard to these aliphatic organochlorine compounds, especially TCE,recently a process for aerobic microbial decomposition has beenreported, more particularly, a biodegradation process in which TCE isepoxidized by an oxygenase-type enzyme (Methylocystis sp. strain Mcontaining methane monooxygenase (Agric. Biol. Chem., 53, p.2903 (1989),Biosci. Biotech. Biochem., 56, p.486 and 56, p.736 (1992)); Methylosinustrichosporium OB3b (Am. Chem. Soc. Natl. Meet. Dev. Environ. Microbiol.,29, p.365 (1989), Appl. Environ. Microbiol., 55, p.3155 (1989), Appl.Biochem. Biotechnol., 28, p.877 (1991)); Pseudomonas putida BHcontaining phenol hydroxylase (Journal of Sewerage Association, 24, p.27(1987)); Acinetobactor sp. strain G4 containing toluene monooxygenase(Appl. Environ. Microbiol., 52, p.383 (1986), 53, p.949 (1987), 54,p.951 (1989), 56, p.279 (1990) and 57, p.193 (1991)); Pseudomonasmendocina KR-1 (Bio/Technol., 7, p.282 (1989)); Pseudomonas putida Flcontaining toluene dioxygenase (Appl. Environ. Microbiol., 54, p.1703(1988) and 54, p.2578 (1988)); Nitrosomonas europaea containing ammoniamonooxygenase (Appl. Environ. Microbiol., 56, p.1169 (1990)).

It is considered that epoxidized TCE by an oxygenase-type enzyme aslisted above is further decomposed biologically or non-biologically, andglyoxylic acid and dichloroacetic acid are produced by the protonizationof TCE epoxide at this stage.

Namely, TCE decomposition by a microorganism containing anoxygenase-type enzyme necessarily produces dichloroacetic acid at acertain ratio. Accordingly, by treating the resulting dichloroaceticacid with a microorganism capable of decomposing halogenated organicacids, TCE can be finally decomposed into substances originally presentin the natural world.

The detailed process of the present invention is: TCE-polluted groundwater or soil is simultaneously or successively treated with two kindsof microorganisms, one of which aerobically converts TCE into TCEepoxide and another one decomposes dichloroacetic acid generated at acertain ratio by the above reaction, into carbon dioxide, water andchloride ion, thus decomposing the soil and water polluting TCE intoharmless substances.

The microorganism used for decomposing TCE in the present invention canbe any microorganism so long as it decomposes TCE through epoxidation ofTCE; therefore all of the above-mentioned TCE-decomposing strains can beused. In addition, those unidentified microorganisms, unisolatedmicroorganisms, a group of symbiotic microorganisms and newly isolatedand identified microorganisms can be also used so long as they have sucha characteristic or activity as mentioned above. Already identifiedmicroorganisms having such a characteristic or activity and applicableto the present invention include bacteria of genus Pseudomonas,Acinetobactor, Xanthobacter and Corynebacterium, particularlyadvantageously used are Pseudomonas cepacia, Pseudomonas putida,Pseudomonas fluorescence and Pseudomonas aeruginosa. For example,Pseudomonas cepacia KK01 (FERM BP-4235; hereafter referred to as strainKK01) which is isolated from the intestine of Nasutitermes takasagoensisor Corynebacterium sp. strain J1 (FERM P-14332; hereafter referred to asstrain J1).

For the microorganism for decomposing halogenated organic acids, anymicroorganism can be used so long as they decompose halogenated organicacids, for example, halogenated acetic acids, particularlydichloroacetic acid into substances originally present in the naturalworld, such as carbon dioxide, water and chloride ion; therefore theabove-mentioned bacteria which decompose dichloroacetic acid can beused. In addition, those unidentified microorganisms, unisolatedmicroorganisms, a group of symbiotic microorganisms and newly isolatedand identified microorganisms can be also used so long as they have sucha characteristic or activity as mentioned above. Already identifiedmicroorganisms having such a characteristic or activity and applicableto the present invention include bacteria of genus Pseudomonas,Acinetobactor, Xanthobacter and Renobacter, particularly advantageouslyused are Pseudomonas putida, Pseudomonas dehalogenns, and Xanthobacterautotrophicus. Renobacter sp. strain AC (FERM BP-5353; hereafterreferred to as strain AC) which is isolated from the Kanto loam soil inJapan, is suitably used in view of its decomposing activity tohalogenated organic acids.

TCE-decomposing process of the present invention is applicable to anyTCE-polluted materials so long as the microorganism can survive. As iseasily understood from the following examples, the cleaning process ofthe present invention is also applicable to various products, forexample, textiles, paper goods and leather goods which can stand thetreatment of relatively short period using an aqueous medium, since thisprocess uses the microorganism in an aqueous medium. An example fordecomposing TCE in soil or ground water and remedying the soil or groundwater, to which the present invention is preferably applicable will nowbe explained. First, TCE is brought into contact with theabove-mentioned bacteria capable of decomposing TCE to formdichloroacetic acid, the resulting dichloroacetic acid is brought intouch with the above-mentioned bacteria capable of decomposingdichloroacetic acid.

Contact between TCE-decomposing bacteria and TCE can be conducted byculturing the microorganism in an aqueous medium containing TCE oradding the aqueous medium and soil to a culture of the microorganism.

Contact between the bacteria capable of decomposing dichloroacetic acidand dichloroacetic acid can be conducted by culturing the microorganismin an aqueous medium in which TCE has already been decomposed byTCE-decomposing bacteria, or adding the aqueous medium and soil to theculture of the microorganism. Various methods such as batch method,semi-continuous method, continuous method and the like can be employedfor this process. The microorganism may be used in a half immobilized orfully immobilized state to a suitable carrier.

The process of the present invention is applicable to treating disposalwater, soil etc. in both closed system and open system. Themicroorganism can be used in an immobilized state on a carrier, or othervarious methods to promoting microbial growth can be concomitantly used.

A number of embodiments of the present invention will now be explainedin more detail with reference to the following examples.

In the following examples, halogenated organic acids were determined byhigh performance liquid chromatography (HPLC) using a column packed withan ion-exchange resin (developed with a solution of 0.01 N sulfuricaqueous solution/acetonitrile=95/5, Detection at 210 nm).

EXAMPLE 1

Decomposition of dichloroacetic acid using Renobacter sp. strain AC

A colony of strain AC grown on the agar medium was inoculated into 100ml of M9 medium containing 0.1% of yeast extract in a 200 ml Sakaguchiflask (a flat bottom flask with shoulder), and cultured with shaking at30° C. for 48 hours. The cells of strain AC grew in an aggregated formuntil about 30 hours, and then started to disintegrate.

Ten samples (Sample Nos. 1-10) were prepared by inoculating 1 ml of theabove culture solution into 50 ml of M9 medium containing dichloroaceticacid at 200 ppm, followed by shaking culture at 30° C. From Sample No.1, one milliliter sample was collected after eight hours of culture andcells was removed by centrifugation, then the supernatant was adjustedto pH 2 or less with dilute sulfuric acid. Then the dichloroacetic acidconcentration was measured by HPLC. From Sample No. 2 after 16 hours,from Sample No. 3 after 24 hours and so on, samples were also taken inthe same manner to determine the dichloroacetic acid concentration everyeight hours, to measure the change of dichloroacetic acid concentrationwith time.

As shown in FIG. 1, decomposition of dichloroacetic acid started after 8hours and dichloroacetic acid of 200 ppm was completely decomposed after48 hours. As an intermediate product, glyoxylic acid was detected, thusconfirming the participation of dehalogenase in decomposition. Glyoxylicacid was completely decomposed into carbon dioxide and water in the end.

EXAMPLE 2

Decomposition of other halogenated acetic acids using Renobacter sp.strain AC

Chloroacetic acid (200 ppm), trichloroacetic acid (50 ppm) andbromoacetic acid (50 ppm) were subjected to decomposition by strain ACin the same manner as in EXAMPLE 1. The relations between culture periodand the concentration of each residual compound are shown in FIGS. 2, 3and 4 respectively.

These compounds were all completely decomposed within 72 hours. As anintermediate product, glyoxylic acid was detected, thus confirming theparticipation of dehalogenase in decomposition. Glyoxylic acid wascompletely decomposed into carbon dioxide and water in the end.

EXAMPLE 3

Decomposition of chloropropionic acid using Renobacter sp. strain AC

2-chloropropionic acid and 3-chloropropionic acid were decomposed bystrain AC in the same manner as in EXAMPLE 1. Concentration of eachcompound was 1000 ppm. The relations between culture period and theresidual concentration of each compound are shown in FIG. 5. In FIG. 5,(1) indicates the residual of 2-chloropropionic acid, and (2) theresidual concentration of 3-chloropropionic acid.

All these compounds were completely decomposed within 48 hours. As anintermediate product, lactic acid was detected, thus confirming theparticipation of dehalogenase in decomposition. Lactic acid wascompletely decomposed into carbon dioxide and water in the end. Thelactic acid was completely decomposed into carbon dioxide and water.

EXAMPLE 4

Decomposition of 2,2-dichloropropionic acid using Renobacter sp. strainAC

2,2-dichloropropionic acid was decomposed using strain AC in the samemanner as in EXAMPLE 1. Concentration of the compound was 100 ppm. Therelations between culture period and the residual concentration of thecompound are shown in FIG. 6.

2,2-dichloropropionic acid (100 ppm) was completely decomposed within 48hours. As an intermediate product, pyruvic acid was detected, thusconfirming the participation of dehalogenase in decomposition. Pyruvicacid was completely decomposed into carbon dioxide and water in the end.

EXAMPLE 5

Remediation of soil polluted with dichloroacetic acid using Renobactersp. strain AC

A colony of strain AC grown on an agar medium was inoculated into 100 mlof M9 medium containing 0.1% yeast extract in a 200 ml Sakaguchi flask,and cultured with shaking at 30° C. for 48 hours. The cells of strain ACgrew in an aggregated form for about 30 hours, and then started todisintegrate.

Ten milliliter of an aqueous solution of dichloroacetic acid was addedto 50 g of air-dried brown forest soil collected in Atsugi-shi,Kanagawaken, Japan to make the dichloroacetic acid concentration 50 mg/gwet soil, to which 10 ml of the above culture solution was mixed andthoroughly stirred, and incubated in a 100 ml conical flask at 30° C.One gram of the soil was taken every 24 hours, to which 5 ml of 0.01 Nsulfuric acid aqueous solution was added and stirred for 1 hour. Thenthe soil was removed by centrifugation and the supernatant was filteredand adjusted to pH 2 or less with dilute sulfuric acid beforeintroducing in HPLC. Thus the daily decrease of dichloroacetic acid wasmeasured. The results are shown in FIG. 7. As a control, an experimentwas conducted using a sterile culture medium instead of cell culture,and the residual rate of dichloroacetic acid was expressed by the ratioof the residual dichloroacetic acid to that in the control experiment.

Decomposition began 2 days after and 50 ppm of dichloroacetic acid wascompletely decomposed after 4 days.

EXAMPLE 6

Remediation of soil polluted with other halogenated organic acids usingRenobacter sp. strain AC

An experiment of soil remediation using strain AC was carried out in thesame manner as in EXAMPLE 5, where the decomposition subjects werechloroacetic acid (50 ppm), trichloroacetic acid (10 ppm) andbromoacetic acid (10 ppm). The relation between culture period and theresidual ratio of these compounds are shown in FIG. 8. In FIG. 8, (1),(2) and (3) indicate residual ratios of chloroacetic acid,trichloroacetic acid and bromoacetic acid, respectively.

Chloroacetic acid was completely decomposed by the third day andtrichloroacetic acid and bromoacetic acid by the fifth day of theexperiment.

EXAMPLE 7

Decomposition of chloropropionic acid in soil using Renobacter sp.strain AC

2-chloropropionic acid and 3-chloropropionic acid were decomposed usingstrain AC in the same manner as in EXAMPLE 5. Concentration of eachcompound was 200 ppm. The relation between culture period and theresidual rate of each compound is shown in FIG. 9. In FIG. 9, (1)represents the residual rate of 2-chloropropionic acid and (2) theresidual rate of 3-chloropropionic acid.

All these compounds were completely decomposed by the third day.

EXAMPLE 8

Decomposition of 2,2-dichloropropionic acid in soil using Renobacter sp.strain AC

2,2-dichloropropionic acid was decomposed using strain AC in the samemanner as in EXAMPLE 5. The concentration of the compound was 50 ppm.The relation between culture period and of residual rate of the compoundis shown in FIG. 10.

The compound was completely decomposed by the third day.

EXAMPLE 9

Decomposition mechanism of each halogenated organic acid usingRenobacter sp. strain AC

One milliliter of the same culture broth as used in EXAMPLE 1 wasinoculated into each 50 ml of the same culture medium containingdichloroacetic acid, chloroacetic acid, trichloroacetic acid,bromoacetic acid, 2-chloropropionic acid, 3-chloropropionic acid or2,2-dichloropropionic acid at the same concentration as in Examples 1-4,respectively, and cultured with shaking at 30° C. At an appropriatetime, when half of the compound was decomposed, 1 ml of each reactionfluid was collected, the cells were removed by centrifugation and pH wasadjusted to pH 2 or less with dilute sulfuric acid. Then the sample wasintroduced into HPLC to analyze the decomposition intermediates. As aresult, formation of following intermediate products were confirmed;glyoxylic acid from dichloroacetic acid, glycolic acid from chloroaceticacid, trichloroacetic acid, and bromoacetic acid, lactic acid from2-chloropropionic acid, and pyruvic acid from 2,2-dichloropropionic acid(no intermediate product from 3-chloropropionic acid), clearly showingthat decomposition of each halogenated organic acid by Renobacter sp. ACstrain was due to the action of dehalogenase, a hydrolyticdehalogenating enzyme. It was confirmed that all the above intermediateproducts were completely decomposed afterwards.

EXAMPLES 10-13 AND COMPARATIVE EXAMPLES 1-3

M9 medium used in Examples 10-13 and Comparative Examples 1-3 has thefollowing composition.

M9 culture medium composition (per one liter)

    ______________________________________                                               Na.sub.2 HPO.sub.4                                                                   6.2          g                                                    KH.sub.2 PO.sub.4         3.0 g                                               NaCl                      0.5 g                                               NH.sub.4 Cl              1.0 g                                                water                    to 1 liter                                           (pH 7.0)                                                                    ______________________________________                                    

Measurement of TCE concentration was all done by head space gaschromatography. Specifically, 5 ml of M9 medium containing a certainconcentration of TCE and 100 μl of cell suspension were put in a 20 mlserum bottle and then the bottle was sealed by a rubber stopper and analuminum cap, and incubated with shaking at 30° C. for a certain periodof time, then 0.1 ml of the gas phase was sampled and measured by gaschromatography. In the examples using soil also, the measurement of TCEconcentration was done according to the above method. Dichloroaceticacid was determined as follows: the reaction mixture was adjusted to pH2 or lower with dilute sulfuric acid and centrifuged. The supernatantwas extracted with diethylether and ethyl acetate, and the extract wasdried to solid using a rotary evaporator and redissolved indemineralized water. The solution was introduced into a high performanceliquid chromatograph (HPLC) with an eluent of 0.01 N aqueous sulfuricacid/acetonitrile=95/5 (v/v). It was confirmed by GC/MS that the peak ofeluate observed at the retention time corresponding to the standarddichloroacetic acid in HPLC, corresponds to dichloroacetic acid. In theexamples using soil, measurement of dichloroacetic acid was also carriedout in the same manner as above except that the soil was directlyextracted with diethylether and ethyl acetate.

In the following Examples 10-13 and Comparative Examples 1-3,Pseudomonas cepacia strain KK01 (FERM BP-4235) and Corynebacterium sp.strain J1 (FERM BP-5102) were used in addition to Renobacter sp. strainAC (FERM BP-5353) mentioned above.

The above three microbial species have been deposited in the followingInternational Deposition Authority.

Name: National Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology

Address: 1--1, Higashi, 1-chome, Tsukuba-shi, Ibaraki-ken, JapanPseudomonas cepacia strain KK01 was initially deposited as FERM P-12869on Mar. 11, 1992 and was transferred to Accession No. FERM BP-4235 onMar. 9, 1993. A complete taxonomic description is found in pendingapplication Ser. No. 08/810,366, filed Mar. 3, 1997. The completetaxonomic description of FERM BP-4235 is as follows:

A. Morphological Properties

(1) Gram stain: Negative

(2) Size and shape of the bacteria: Bacillus having a length of 1.0-2.0μm and a width of about 0.5 μm.

(3) Mobility: Present

B. Growth state of the bacteria in each culture medium.

    ______________________________________                                                           Culture   Growth                                             Culture Medium                    Temp. (° C.)     State             ______________________________________                                        Blood agar culture medium                                                                        37        +                                                  Lactose agar culture medium           37           +                          Chocolate agar culture medium         37           ++                         GMA                                  37           -                           Scyllo                               37            -                          Usual agar culture medium             4             -                         Usual agar culture medium            25            ±                       Usual agar culture medium            37            -                          Usual agar culture medium            41            ±                     ______________________________________                                    

C. Physiological properties

(1) Aerobic or anaerobic: Strictly aerobic

(2) Degradation type of saccharose: Oxidation type

(3) Production of oxidase: +

(4) Reduction of silver nitrate: +

(5) Production of hydrogen sulfide: -

(6) Production of indole: -

(7) Production of urease: -

(8) Liquefaction of gelatin: -

(9) Hydrolysis of arginine: -

(10) Decarboxylation of lysine: +

(11) Decarboxylation of ornithine: +

(12) Utilization of citric acid: +

(13) Methylcarbinolacetyl reaction (VP reaction): -

(14) Detection of tryptophane deaminase: -

(15) ONPG: -

(16) Utilization of carbohydrates:

Glucose: +

Fruit Sugar: +

Maltose: +

Galactose: +

Xylose: +

Mannitol: +

White Sugar: -

Lactose: +

Aesculin: -

Inositol: -

Sorbitol: -

Rhamnose: -

Melibiose: -

Amygdalin: +

L-(+)-arabinose: +

Corynebacterium sp. J1 was originally deposited as FERM P-14332 on May25, 1994 and later transferred to Accession No. FERM BP-5102 on May 17,1995. A complete taxonomic description is found in allowed applicationSer. No. 08/454,515, filed May 30, 1995, now U.S. Pat. No. 5,807,736,issued Sep. 15, 1998. Renobacter sp. strain AC was originally depositedunder Accession No. FERM P-14641 on Nov. 15, 1994 and was transferred toAccession No. FERM BP-5353 on Dec. 25, 1995.

The complete taxonomic description of FERM BP-5102 is as follows:

Gram straining and morphology: Gram-negative rod

Growth condition in each medium

BHIA: good

MacConkey: possible

Color of colony: cream

Optimum growth temperature: 25° C.>30° C.>35° C.

Motility: negative (in semisolid medium)

TSI (slant/butt): alkali/alkali, H₂ S(-)

Oxidase: positive (weak)

Catalase: positive

Fermentation of sugars

glucose: negative

sucrose: negative

raffinose: negative

galactose: negative

maltose: negative

Urease: positive

Esculin hydrolysis (β-glucosidase): positive

Nitrate reduction: negative

Indole productivity: negative

Glucose acidification: negative

Arginine dihydrase: negative

Gelatin hydrolysis (protease): negative

β-galactosidase: negative

Assimilation of each compound

glucose: negative

L-arabinose: negative

D-mannose: negative

D-mannitol: negative

N-acetyl-D-glucosamine: negative

maltose: negative

potassium gluconate: negative

n-capric acid: positive

adipic acid: negative

dl-malic acid: positive

sodium citrate: positive

phenyl acetate: negative

It is evident that TCE-decomposing strains KK01 and J1 have oxygenase,because they produce 2-hydroxymuconic acid semialdehyde which hasmaximum absorption at 375 nm as an intermediate product of phenoldecomposition. It has been confirmed that this oxygenase participates indecomposition of TCE.

It have been also confirmed from above Examples 1-9 that decompositionof dichloroacetic acid by strain AC was due to the action ofdehalogenase, a hydrolytic dehalogenating enzyme.

EXAMPLE 10

Decomposition of TCE and dichloroacetic acid using mixed culture systemof strains KK01 and strain AC

A colony of strain AC grown on an agar medium was inoculated into 100 mlof M9 medium containing 0.1% of yeast extract in a 200 ml Sakaguchiflask, and cultured with shaking at 30° C. for 48 hours. Then a colonyof strain KK01 on an agar medium was inoculated into 100 ml of M9 mediumcontaining 0.1% of yeast extract and 200 ppm of phenol in another 200 mlSakaguchi flask, and cultured with shaking at 30° C. for 18 hours.

Then the both cells were collected and 100 μl was added to 5 ml of M9medium containing 10 ppm of TCE in a vial to the each cell density of6-8×10⁸ cells/ml. The vial was sealed with a rubber stopper and analuminum cap and cultured with shaking at 30° C. Measurements of TCEconcentration were made every 6 hours in the same manner as mentionedabove. At the same time, a suspension of the above two strains was addedto 1 liter of M9 medium containing 10 ppm TCE in a Sakaguchi flask to acell concentration of 6-8×10⁸ cells/ml and cultured with shaking at 30°C. The dichloroacetic acid concentration was determined every 6 hours inthe same manner as mentioned above.

The results are shown in FIG. 11. Dichloroacetic acid concentration isexpressed as a concentration in the original culture. In FIG. 11, (1)indicates the residual concentration of TCE and (2) indicates theresidual concentration of dichloroacetic acid.

It was shown that dichloroacetic acid was formed accompanying thedecomposition of TCE which was completely decomposed within 16 hours,and decomposed completely within 36 hours.

COMPARATIVE EXAMPLE 1

Decomposition of TCE using strain KK01 and formation of dichloroaceticacid

Concentration of TCE and dichloroacetic acid were measured in the samemanner as in EXAMPLE 10 except that only strain KK01 was used.

The results are shown in FIG. 12. In FIG. 12, the numeral (1) indicatesresidual concentration of TCE and the numeral (2) indicates that ofdichloroacetic acid.

It was revealed that decomposition rate of TCE was a little higher thanin a mixed system of strain AC and strain KK01, but that about 700 ppbof dichloroacetic acid remained undecomposed.

EXAMPLE 11

Decomposition of TCE and dichloroacetic acid in mixed culture system ofstrain J1 and strain AC

Strain J1 and strain AC were inoculated in the same manner as in EXAMPLE10 and the concentrations of TCE and dichloroacetic acid were measured.The results are shown in FIG. 13. In FIG. 13, the numeral (1) indicatesresidual concentration of TCE and the numeral (2) indicates that ofdichloroacetic acid.

Dichloroacetic acid was formed accompanying the decomposition of TCEwhich was completely decomposed in 16 hours, and completely decomposedwithin 30 hours.

COMPARATIVE EXAMPLE 2

Decomposition of TCE using strain J1 and formation of dichloroaceticacid

The concentrations of TCE and dichloroacetic acid were measured in thesame manner as in Example 10 except that only strain J1 was used.

The results are shown in FIG. 14. In FIG. 14, the numeral (1) indicatesresidual concentration of TCE and the numeral (2) indicates that ofdichloroacetic acid.

It was revealed that decomposition rate of TCE was a little higher thanin a mixed system of strain AC and strain J1, but that about 350 ppb ofdichloroacetic acid remained undecomposed.

EXAMPLE 12

Decomposition of TCE and dichloroacetic acid in soil in mixed culturesystem of strain J1 and strain AC

Five grams of air-dried brown forest soil collected in Atsugi-shi,Kanagawa-ken, Japan was put in a 20 ml serum bottle and 1 ml of a TCEaqueous solution was added to make the TCE concentration 10 mg TCE/g wetsoil. Then the cell suspensions of strain J1 and strain AC prepared inEXAMPLE 11 were inoculated into the soil to each cell concentration of6-8×10⁸ cells/g wet soil, and the bottle was sealed with an aluminum capand incubated at 30° C. The daily change of TCE concentration wasdetermined in the same manner as above. At the same time, 10 ml of anaqueous TCE solution was added to 50 g of air-dried brown forest soilcollected in Atsugi-shi, Kanagawa-ken, Japan in make the TCEconcentration 10 mg TCE/g wet soil. Then the cells of strain J1 andstrain AC prepared in EXAMPLE 11 were inoculated into the soil to eachcell concentration of 6-8×10⁸ cells/g wet soil, and the soil wasincubated at 30° C. in an 100 ml serum bottle having a screw cap linedwith Teflon liner. One gram of the soil was taken every 24 hours andstirred for one hour in 5 ml of 0.01 N aqueous sulfuric acid, and thenthe soil was removed by centrifugation and filtration and the resultedliquid was adjusted to pH 2 or below with dilute sulfuric acid andintroduced in HPLC. Thus the daily decrease of dichloroacetic acid wasmeasured. The results are shown in FIG. 15. In FIG. 15, the numeral (1)indicates the residual concentration of TCE and the numeral (2) that ofdichloroacetic acid.

It was revealed that on the first day the concentration ofdichloroacetic acid increased temporarily as TCE was decomposed, butdichloroacetic acid began to undergo decomposition after that and bothTCE and dichloroacetic acid were completely decomposed on the fourthday.

COMPARATIVE EXAMPLE 3

Decomposition of TCE in soil using strain J1 and formation ofdichloroacetic acid

The concentrations of TCE and dichloroacetic acid were measured in thesame manner as in Example 12 except that only strain J1 was used. Theresults are shown in FIG. 16. In FIG. 16, the numeral (1) indicatesresidual concentration of TCE and the numeral (2) indicates that ofdichloroacetic acid.

It was revealed that decomposition rate of TCE was a little higher thanin a mixed system of strain AC and strain J1, but dichloroacetic acidremained at about 230 μg/kg wet soil without decomposition.

As described above, the present invention provides a new microbialstrain capable of decomposing halogenated organic acids and a processfor decomposing halogenated organic acids which is one of the currentproblems, thus enabling the efficient microbial treatment of the liquidwastes containing halogenated organic acids as well as the soil pollutedwith halogenated organic acids.

Also according to the present invention, aliphatic organochlorinecompounds, particularly trichloroethylene, can be substantiallycompletely decomposed into substances originally present in the naturalworld, and soil polluted with aliphatic organochlorine compounds can beremedied well.

What is claimed is:
 1. A biologically pure culture of Renobacter sp.FERM BP-5353 capable of decomposing halogenated organic acid.
 2. Aprocess for remedying environment contaminated with an aliphaticorganochlorine compound comprising the steps of:providing first andsecond microorganisms which are different from each other, the firstmicroorganism is Pseudomonas cepacia strain KK01 (FERM BP-74235) orCorynebacterium sp. (FERM BP-5102) and capable of introducing an oxygenatom into the aliphatic organochlorine compound to convert the aliphaticcompound to an epoxide thereof, and the second microorganism isRenobacter sp. (FERM BP-5353) and capable of decomposing a halogenatedorganic acid to substances naturally existing in nature; and introducingthe first and second microorganisms into the environment, wherein thefirst microorganism converts the aliphatic organochlorine compound intoan epoxide thereof, said epoxide being protonized during decompositionthereof to produce a chlorinated organic acid; and wherein the secondmicroorganism converts the chlorinated organic acid into substancesnaturally existing in nature.
 3. The process according to claim 2,wherein the aliphatic organochlorine compound is trichloroethylene.